APPLICANT?S DESCRIPTION: The long term objective of this study is to determine how signal transduction pathways in the nucleus regulate chromosomal architecture and gene expression. Towards this end, we have identified a novel tandem kinase in Drosophila, JIL-1, that associates with the chromosomes throughout the cell cycle, localizes specifically to the gene-active interband regions of the larval polytene chromosomes, is capable of phosphorylating histone H3 in vitro, and is enriched almost two-fold on the transcriptionally hyperactive male larval polytene X chromosome due to its association with the MSL dosage compensation complex. Histone H3 serine 10 phosphorylation levels are severely reduced in embryos from a hypomorphic JIL-1 mutant, thus placing JIL-l in the pathway mediating histone H3 phosphorylation. In addition, with high penetrance these mutants (which contain low levels of JIL-l kinase) show a number of chromosomal abnormalities, including lack of condensation at mitosis, disorganization in alignment at the mitotic spindle, separation from centrosomes, and ultimately chromosomal breakdown and fragmentation. Only 5 percent of the embryos survive to hatching, and of the animals that survive to adulthood, females outnumber males by a 2:1 ratio, supporting the hypothesis that JIL-1 is essential in dosage compensation mechanisms. Thus, these findings suggest a model where JIL-l signaling plays a direct functional role in chromosomal regulation and maintenance throughout the cell cycle, including condensation and segregation of chromosomes at metaphase as well as modification of chromatin and transcriptional regulation at interphase. We propose to test this model by generating a range of JIL-l mutant alleles, including a complete null, which will allow us to genetically dissect the functional requirements for JIL-l in different pathways and at different stages. We will determine whether histone H3 is a direct or indirect target of JIL-1 using biochemical approaches, and we will identify other potential substrates and interaction partners using genetic and yeast two-hybrid approaches. Our expectation is that JIL-l function is necessary in more than one chromatin remodeling complex, since JIL-l is found in females and on autosomes in addition to its presence on the male X-specific chromatin remodeling complex. The powerful molecular, genetic, and cellular approaches available in Drosophila are uniquely suited to the analysis of such complex signaling pathways. Thus, these studies will provide valuable new insights into the role of phosphorylation in regulation of gene expression and chromatin structure of fundamental significance for understanding both normal and cancerous developmental processes.